Molecular basis for thermal stability and affinity in a VHH: Contribution of the framework region and its influence in the conformation of the CDR3

Abstract The camelid single domain antibody, referred to VHH or Nanobody, is considered a versatile tool for various biotechnological and clinical applications because of its favorable biophysical properties. To take advantage of these characteristics and for its application in biotechnology and therapy, research on VHH engineering is currently vigorously conducted. To humanize a camelid VHH, we performed complementarity determining region (CDR) grafting using a humanized VHH currently in clinical trials, and investigated the effects of these changes on the biophysical properties of the resulting VHH. The chimeric VHH exhibited a significant decrease in affinity and thermal stability and a large conformational change in the CDR3. To elucidate the molecular basis for these changes, we performed mutational analyses on the framework regions revealing the contribution of individual residues within the framework region. It is demonstrated that the mutations resulted in the loss of affinity and lower thermal stability, revealing the significance of bulky residues in the vicinity of the CDR3, and the importance of intramolecular interactions between the CDR3 and the framework‐2 region. Subsequently, we performed back‐mutational analyses on the chimeric VHH. Back‐mutations resulted in an increase of the thermal stability and affinity. These data suggested that back‐mutations restored the intramolecular interactions, and proper positioning and/or dynamics of the CDR3, resulting in the gain of thermal stability and affinity. These observations revealed the molecular contribution of the framework region on VHHs and further designability of the framework region of VHHs without modifying the CDRs.


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Page 2 Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 7XL0 1 Overall quality at a glance i ○ The following experimental techniques were used to determine the structure:

X-RAY DIFFRACTION
The reported resolution of this entry is 1.70 Å.
Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic. The table shows the number of entries on which the scores are based. The table below summarises the geometric issues observed across the polymeric chains and their fit to the electron density. The red, orange, yellow and green segments of the lower bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria respectively. A grey segment represents the fraction of residues that are not modelled. The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5% The upper red bar (where present) indicates the fraction of residues that have poor fit to the electron density. The numeric value is given above the bar.

Mol Chain Length
Quality of chain 2 Entry composition i ○ There are 4 unique types of molecules in this entry. The entry contains 2065 atoms, of which 0 are hydrogens and 0 are deuteriums.
In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.
• Molecule 1 is a protein called Nanobody Vobarilizumab.   3 Residue-property plots i ○ These plots are drawn for all protein, RNA, DNA and oligosaccharide chains in the entry. The first graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic. The second graphic shows the sequence view annotated by issues in geometry and electron density. Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more. A red dot above a residue indicates a poor fit to the electron density (RSRZ > 2). Stretches of 2 or more consecutive residues without any outlier are shown as a green connector. Residues present in the sample, but not in the model, are shown in grey.

Standard geometry i ○
Bond lengths and bond angles in the following residue types are not validated in this section: GOL, SO4 The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value. A bond length (or angle) with |Z| > 5 is considered an outlier worth inspection. RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles). There are no bond angle outliers.

Mol Chain
There are no chirality outliers.
There are no planarity outliers. The all-atom clashscore is defined as the number of clashes found per 1000 atoms (including hydrogen atoms). The all-atom clashscore for this structure is 5.

Too-close contacts
All (19)  There are no symmetry-related clashes.

Protein backbone i ○
In the following table, the Percentiles column shows the percent Ramachandran outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the backbone conformation was analysed, and the total number of residues. In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.

Mol Chain
The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues.  Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 7XL0

Carbohydrates i ○
There are no monosaccharides in this entry.
5.6 Ligand geometry i ○ 3 ligands are modelled in this entry.
In the following table, the Counts columns list the number of bonds (or angles) for which Mogul statistics could be retrieved, the number of bonds (or angles) that are observed in the model and the number of bonds (or angles) that are defined in the Chemical Component Dictionary. The Link column lists molecule types, if any, to which the group is linked. The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value. A bond length (or angle) with |Z| > 2 is considered an outlier worth inspection. RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles). There are no bond angle outliers.

Mol Type Chain Res Link
There are no chirality outliers.
There are no torsion outliers.
There are no ring outliers.
No monomer is involved in short contacts.

Other polymers i ○
There are no such residues in this entry. Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 7XL0

Polymer linkage issues i ○
There are no chain breaks in this entry.
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Page 12 Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 7XL0 6 Fit of model and data i ○ 6.1 Protein, DNA and RNA chains i ○ In the following table, the column labelled '#RSRZ> 2' contains the number (and percentage) of RSRZ outliers, followed by percent RSRZ outliers for the chain as percentile scores relative to all X-ray entries and entries of similar resolution. The OWAB column contains the minimum, median, 95 th percentile and maximum values of the occupancy-weighted average B-factor per residue. The column labelled 'Q< 0.9' lists the number of (and percentage) of residues with an average occupancy less than 0.9. 6.2 Non-standard residues in protein, DNA, RNA chains i ○ There are no non-standard protein/DNA/RNA residues in this entry.

Carbohydrates i ○
There are no monosaccharides in this entry. Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 7XL0 median, 95 th percentile and maximum values of B factors of atoms in the group. The column labelled 'Q< 0.9' lists the number of atoms with occupancy less than 0.9.